This research aims to uncover novel immune system components either unique to the trout, or present in human immune system pathways in ways that could not have been anticipated. Specifically, it aims to characterize the similarities and differences in immune progenitors between two lines of rainbow trout, a line resistant to the fish pathogen Flavobacterium psychrophilum (Fp) which causes Bacterial Cold Water Disease, and a susceptible (control) line. The presence of blood progenitor cells has long been observed in humans and provides an important source of immune cells for transplantation. A recent discovery by the PI suggests that trout blood contains abundant progenitor cells. Because trout have much higher densities of blood PBLs compared to humans, and further, possess higher percentages of these progenitors in their wbcs, rainbow trout provides an excellent animal model to further characterize their phenotype and responsiveness to bacterial pathogens. In this regard, the PI recently discovered that Fp-resistant trout have a significantly higher abundance of progenitors compared to Fp-susceptible trout. This suggests that progenitors may play a role in conferring resistance to Bacterial Cold Water Disease. AIM 1 will compare cell abundance of B/myeloid and myeloid/granulocyte progenitors between Fp-resistant and Fp-susceptible trout, using flow cytometry. Further, immunoglobulin rearrangement for heavy and light chains will be determined in granulocyte/myeloid cells using PCR, to test the hypothesis that Fp-resistant fish have a higher capacity to shift B lineage cells towards the myeloid lineage. In AIM 2, the phagocytic nature of progenitors will be determined in PBLs, spleen and kidney using fluorescently labeled Fp bacteria and analyzed by flow cytometry and confocal microscopy. Further, Fp-resistant and susceptible lines of trout will be exposed to Fp in challenge experiments performed with undergraduate students at the National Center for Cool and Cold Water Aquaculture in collaboration with Dr. Greg Wiens; phagocytic characteristics (onset, frequency) will be compared between the two lines of trout, and correlated with survival. In AIM 3, it will be determined how in vivo LPS-induction affects the abundance of progenitors in blood and how this drives their developmental fate. Lastly, it will be explored if and how in vivo injection of the CXCR4-antagonist Plerixafor increases the abundance of progenitor cells in trout blood, and if so, if this increases survival of susceptible fish after Fp-challenge. Together, thes studies should increase understanding of the diverse ways progenitor cells respond to pathogenic threats, and may lead to novel approaches in the treatment of human bacterial infections.